Production of riboflavin by fermentation processes



Patented Sept. 14, 1948 PRODUCTION OF RIBOFLAVIN BY FER- MENTATIONPROCESSES Henry L. Pollard, Nelson E. Rodgers, and Reginald E. Meade,Appleton, Wls., assignors to Western Condensing Company, San Francisco,Calif., a corporation of California No Drawing. Application November 11,1944, Serial N 563,085

2 Claims. (01.195-42) l 2 This invention relates to processes for theThe thus prepared material, cooled to a temmanui'acture of biologicallyactive materials such perature of about 100 F., is placed in a fermentasvitamins by fermentation processes. More ing container and inoculatedwith Clostridium particularly, the invention pertains to the syn--acetobutylicmn (such as described by McCoy, thesis, fromlactose-containing products (in par- 5 t rson and Ha in s n u n l of Infctious ticular, lacteal material), of riboflavin and other sea es." Voume P e 457) Pre r b y at a vitamins by the action of bacteria, and,speciftemperature about 100 F. under conditions such ically, by theaction of the bacterium Clostridium as will prevent the introduction ofiron and conacetobutylicum. taminating organisms. A suitable inoculum.may

The present application is a continuation-ine prepared from a stockculture by repeated part of the application Serial No. 439,310, filedtransfers to a nutrient medium such as whey. April 1'7, 1942, by HenryL. Pollard, Nelson E. In n ral. fe m ntat on f a bat or w y Rodgers andReginald E. Meade, entitled "Process can continue for from twelve toforty-eight hours, for manufacturing a vitamin concentrate (now or untilthere is no noticeable further increase issued as United States PatentNo. 2,369,680) in the riboflavin content.

The following paragraphs describe generall a, The gasesformed duringfermentation can be fermentation process to the improvement of ve ted fom th fermenting a k s o m which the present invention is particularlydi- The solvents formed during fermentation can be rooted. removed byfractional distillation, and after re- As disclosed in our copendingapplication, we moving volatile products the fermented material havefound that the natural riboflavin content of can be concentrated byevaporati n t0 Dmdllce whey or skim milk may be increased to a conaconcentrated liquor. If desired, this liquor siderable extent bysubjecting whey or skim milk can be further subjected to drying toproduce a under controlled conditions to the fermenting powderedproduct. action of Clostridium acetobutylicum. Such a 2 If desired,instead of separately fractionating fermentation synthesis of riboflavinis accomthe solvents, they can be condensed from the panied by theformation of neutral solvents such vapors evolved during concentration01' the feras ethanol, acetone and butanol, and gases such mentedmaterial by evaporation to form a wateras hydrogen and carbon dioxide,which can be solvent mixture from which the solvents can be recovered asvaluable lay-products. removed by fractional distillation.

To prepare a lactose-containing lacteal me- At some point afterfermentation, it is desirdium such as whey or skim milk for fermenableto inhibit further bacterial action, as for tation to increase itsriboflavin content, it is example by heat s eriliz tion pplie as apsterilized completely or substantially completely, arate step or inconjunction with concentraas by heat treatment at' about 250 F. forabout 5 tion by evaporation. 10 to 20' minutes. In addition, the acidityof The produ t o t ed y e above Procedure the lacteal medium isneutralized preferably to is a concentrate which can be further refineda pH of 6 to 7 by adding an alkaline reagent or blended with variousfood material for human such assodium, potassium or calcium hydroxide.or animal consumption. By use of the process Calcium carbonate may beadded t enhance 40 described hereinabove, the riboflavin content ofriboflavin production. The iron content of the w h s en increased f omout .4-t o lacteal medium preferably is adjusted to the 60 to 70micrograms p r m llili er fo e Bonrange of from 0.5 to less than 4.5parts per milcentration), which corresponds to about 240 to lion. Inthis connection it should be noted that 28 0 cr rams r ra n a d d bsisthe natural iron content of uncontaminated whey Some of the lactoseis consumed in the feror skim milk will range from 0.10 to 0.21 partmenting process so that the final product conper million, while the ironcontent of whey contains a reduced amount of milk sug r; p n ntaminated, as by corrosive contact with iron upon the extent offermentation. The solids of containers, may reach a value above 4.5parts the final product are in the remaining solids of per million. Inthe case of uncontaminated the whey or s m mi emp oyed and thereforewhey, the iron content may be adjusted upwardare available as foodingredients which are used 1y by incorporation of suitable amounts of a501- to advantage when the product is blended with uble ferrous saltsuch as ferrous sulfate, while other material, such as various milkproducts, whey containing too much iron may be diluted bread and bakeryproducts, poultry and animal with uncontaminated whey. feeds, and thelike.

The present invention pertains particularly to the addition of solublemagnesium salts to a lactose-containing material to be used in afermenta tion process such as that described hereinabove.

More particularly, we have found that increased riboflavin yields can beobtained by incorporating soluble magnesium salts, such as magnesiumsulfateor magnesium chloride, with whey being fermented.

It is therefore an important object of the present invention to providean improved method for synthesizing riboflavin from whey or the like byfermentation with Clostridium acetobutylicum.

Another important object of the present invention is to provide aprocess of the nature indicated including the step of incorporating asoluble salt of magnesium with whey or other lactosecontaining medium.

Other and further objects and features of the present invention willbecome apparent from the following description and appended claims.

In proceeding according to the present invention, the directions givenhereinabove as applying to the method of the copending application arefollowed, except for the variations disclosed hereinbelow.

The effectiveness of the methods of this invention depends largely uponadding the magnesium salt before sterilizing the medium. Thispoint isdemonstrated in the following experiment, in which riboflavin synthesisin a whey medium supplemented with magnesium sulfate prior tosterilization has been compared with synthesis in a medium to which themagnesium salt was added after sterilization.

The basal medium used inthi experiment was rennet whey supplemented with4.8 parts per million zinc sulfate heptahydrate, parts per billionpara-aminobenzoic acid and 0.2% calcium carbonate. The activity of themagnesium sulfate was tested in the presence of different concentrationsof iron, added as ferrous sulfate, as indicated in the'accompanyingtable. At each of the concentrations indicated the magnesium sulfate wassterilized with the medium in one series of cultures and addedaseptically as a sterile stock solution to the sterilized medium inanother series.

The various media were autoclaved in 100 milliliter volumes, inoculatedwith 4% of a suitable Clostridium acetobutylicum starter and incubatedat 100 F. for 38 hours. The riboflavin yields in duplicate cultures areshownin the fol-. lowing table:

siderably in the presence of all concentrations of Per Cent Flaving./ml., added Fe in parts per million Anhydrous o 8.0 13 49 41 42 421s 1. 7 17 20 as 41 40 15 0 03 29 40 as 40 as 41 13 2s 49 40 49 51 40 12o 06 as 01 as 51 a1 30 13 20 as 02 41 so 40 1s 0 12 so 04 51 50 as 8.120 01 5o 50 50 a9 3. 7 0 30 42 c7 52 4o 40 32 e. 1 47 e0 01 a2 45 a3 4.s o 60 40 e7 49 4s 44 25 3.7 41 c5 49 44 44 21 10 As was observed in thepreceding experiment, the response of riboflavin production to magnesiumsulfate was dependent on the iron concentration. Although the maximumyields of riboflavin were enhanced materially by concentrations ofmagnesium sulfate up to 0.6%, the iron concentrations supporting themaximum yields were reduced considerably. In the presence of lowconcentrations of iron, riboflavin synthesiswas improved throughout theentire.

range of magnesium concentrations tested. In the intermediate range ofiron levels (1.12 to 1.68 points per million) magnesium sulfate up to anoptimum concentration progressively stimulated synthesis, but above thisoptimum concentration, stimulation of synthesis was less marked.

In the presence of added iron in excess of 1.68

parts per million,'synthesis was reduced by magnesium sulfate inconcentrations greater than In general, increased yields of riboflavinare obtained by the addition of from 0.003 to 0.12 or 0.2% of moremagnesium in the form of a soluble magnesium salt such as magnesiumsulfate or chloride. When the iron concentration exceeds about 2 partsper million, the added magnesium should not exceed 0.012%.

The above examples have been given solely Per cent Mgso Addition Flaving.lml., added Fe in parts per million Anhydrous. Before or After MKISOsmmmim o 0.28 0.50 0.84 1.21 1.40 1.08 2.24 a 30 Before-.. 4. 7 4. 7 7.723 48 45 42 31 15.0 0.03 4. 7 4. 5 8.0 22 45 45 41 32 8. 0 After 4. 1 4.3 5. 7 15 20 45 49 35 18. 0 4. 0 4. 0 5. 5 19 29 45 47 36 17. 0 Before5. 0 6. 0 19. 0 56 51 48 41 31 9. 3 0. 06 4. 7 6. 0 19. 0 55 51 49 42 328. 3 After 4. 0 4. 0 6. 0 10 31 47 47 37 16. 0 4. 0 4. 3 0. 0 17 33 4147 38 10. 0 Before; 5. 0 8. 0 27. 0 51 40 47 41 30 6. 0 0. 12 4. 7 6. 629. 0 51 52 47 42 30 6. 5 After 4.0 4. 3 6. 7 16 30 45 47 38 17. 0 4. 04. 3 5. 3 15 38 49 47 38 16. 0

It is evident that supplementing the medium with magnesium sulfate priorto sterilization greatly improved'the maximum riboflavin yields overthose obtained when adding the supplement after sterilization. Also, itis noteworthy that the optimum iron concentration was reduced con- I asillustrative of the applications of the methods of this invention to thesynthesis of riboflavin from lacteal or other lactose-containingliquids. A number of experiments of this general type form the basis forthe claimed methods. Isolated experiments may show minor deviation fromthe may be varied within a wide range without departing from theprinciples of this invention and it is therefore not our purpose tolimit the scope of the patent granted on this inventid'n otherwise thannecessitated by the scope of the appended claims.

We claim as our invention:

1. In the process for manufacturing a vitamin concentrate includingriboflavin by fermenting with C stridium acetobutylicum a materialselected tom the group consisting of whey and skim mills the improvementcomprising incorporating with said material from 0.003 to 0.2% magnesiuminthe form of a soluble magnesium salt, the iron content of saidmaterial in excess of the 0.10 to 0.21 parts per million natural ironcontent of said material being kept within the range of 0.5 to 4.5 partsper million'and not higher than 2 parts per million when at least 0.12%magnesium is added, thereafter sterilizing said material and inoculatingsaid material with a culture of C'lostridium acetobutylicum.

2. In the process of manufacturing a vitamin' concentrate includingriboflavin by fermenting whey with Clostridium acetobutylicum, theimprovement comprising incorporating with said whey from 0.015 to 1%magnesium sulfate. the

iron content of said whey in excess of the 0.10 to 0.21 partsper millionnatural iron content of said whey being kept not lower than 0.5 partsper million and not higher than 2 parts per million, sterilizing saidwhey and inoculating said whey with a culture of Clostridium acetobutulicum.

HENRY L. POLLARD.

NELSON E. RODGERS. 'REGINA LD E. MEADE.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS OTHER REFERENCES Indus. 8; Eng. Chem, Sept. 1930,page 1011.

Porter, Bacterial Chemistry and Physiology, 1946,,Wiley. DD. 629, 674.

Buchanan, Bacteriology, 1938, Macmillan, page 189.

Winton, Structure and Composition of Foods, vol. III, 1937, page 143.

